This year we continue biochemical and biophysical studies on human tyrosinase and mutant variants involved in oculocutaneous albinism OCA1B. We also published our method on thermodynamic analysis of weak protein interactions using sedimentation equilibrium and use molecular modeling to build structures of multi-domain proteins, simulate the effect of pathogenic missense changes, and provide a quantitative analysis of their impact on protein structure and stability for proteins involved in inherited eye disease. Oculocutaneous albinism is a rare genetic disorder of melanin synthesis that results in hypopigmented hair, skin, and eyes. Tyrosinase (TYR) catalyzes the rate-limiting, first step in melanin production and its gene (TYR) is mutated in many cases of oculocutaneous albinism (OCA1), an autosomal recessive cause of childhood blindness. Patients with reduced TYR activity are classified as OCA1B; some OCA1B mutations are temperature-sensitive. Therapeutic research for OCA1 has been hindered, in part, by the absence of purified, active, recombinant wild-type and mutant human enzymes. Currently there is no biologically active human recombinant tyrosinase available in preparative quantities. Therefore we were interested to have an active human protein and OCA1B-related mutant variants expressed and purified for biochemical and biophysical studies to study their enzymatic properties, thermal sensitivity, and structural properties. The intra-melanosomal domain of human tyrosinase (residues 19469) and two OCA1B related temperature-sensitive mutants, R422Q and R422W were expressed in insect cells and produced in T. ni larvae (Dolinska et al., PlosOne, 2014). The short trans-membrane fragment was deleted to avoid potential protein insolubility, while preserving all other functional features of the enzymes. Purified tyrosinase was obtained with a yield of .1 mg per 10 g of larval biomass. The protein was a monomeric glycoenzyme with maximum enzyme activity at 37oC and neutral pH. According to mass-spectroscopy analysis glycosylation occurred at five Asn residues, and the enzymes kinetic and pharmacologic properties were similar to the authentic enzyme described in the literature. The two purified mutants when compared to the wild-type protein were less active and temperature sensitive. These differences are associated with conformational perturbations in secondary structure. The intra-melanosomal domains of recombinant wild-type and mutant human tyrosinases are soluble monomeric glycoproteins with activities which mirror their in-vivo function. This advance allows for the structure function analyses of different mutant TYR proteins and correlation with their corresponding human phenotypes; it also provides an important tool to discover drugs that may improve tyrosinase activity and treat OCA1B. Recently we were invited to submit a manuscript on a method on thermodynamic analysis of weak protein interactions using sedimentation equilibrium. This paper was recently published in Current Protocols in Protein Science (Sergeev et al, 2014). Thermodynamics experiments are necessary to find Gibbs free energy, enthalpy, and entropy and other thermodynamics parameters. However traditional calorimetry is not a sensitive enough method to measure weak interactions between protein molecules and require large protein samples. Our method is novel and based on analytical ultracentrifugation and require a direct determination of the dissociation constant (Kd) for protein-protein interactions as a function of temperature. The dissociation constant, which characterizes the strength of protein association in oligomeric complexes formed by weak forces, can be precisely measured by sedimentation equilibrium. The Kd is dependent on local protein interactions and the temperature. Proteins self-associate to form dimers and tetramers. Purified proteins are used to study the thermodynamics of protein interactions using the analytical ultracentrifuge. In this approach, monomer-dimer equilibrium constants are directly measured at various temperatures. Data analysis is used to derive thermodynamic parameters, such as Gibbs free energy, enthalpy, and entropy, which can predict which weak forces are involved in protein association. Lately we were using molecular modeling to investigate the potential structural and functional consequences as well as possible risks associated with genetic mutations affecting proteins composed of numerous repetitive domains. Mutations in such proteins often cause the inherited eye disease. However computational methods for the analysis of such structures are not establishes yet. These proteins are very large molecules difficult for the structural and computational analysis. However such an analysis could be performed using isolated structural domains. In first case, the analysis of genetic mutations has been implied to the multi-domain FBN-2 protein associated with macular degeneration and AMD phenotypes (Ratnapriya et al., Human Molecular Genetics, 2014). The fibrillin-2 protein, FBN2, is a 2,912 amino acid polypeptide which has one amino-terminal trans-membrane domain, 4 epidermal growth factor-like (EGF) domains, 43 calcium-binding consensus sequences (Ca_EGF domains), and 9 transforming growth factor &#946;1 binding protein-like (TB) domains. FBN-2 has 363 cysteine residues. We have analyzed several mutations in the computationally isolated Ca_EGF domains and structural consequences of these changes. Mutations with severe phenotype are likely to cause a change in the fiber flexibility, packaging, glycosylation pattern, and pathogenicity. Similar approach in the analysis of mutant variants have been applied to novel mutation in fibrillin-1 protein, FBN1, causes ectopia lentis and varicose great saphenous vein in one Chinese autosomal dominant family (Fu et al., Molecular Vision, 2014). We have also participated in collaborative work on the role of chaperone-like molecules in the pathogenesis of inherited eye disease (Valapala M. et al., Autophagy, 2014), the role of IL27 p28 in autoimmunity (Chong WP et al., J. Autoimmunity, 2014), and the biochemical characterization of Interleukin-35 which induces regulatory B cells suppressing autoimmune disease (Wang R.X. et al., Nature Medicine, 2014).